The present invention relates to a method for identifying enzymes and inhibitors of enzymes involved in nucleic acid modification, and particularly a homogenous assay for identifying nucleic acid ligases and helicases and inhibitors thereof using fluorescence polarization to detect the change in mass of a fluorescently labeled oligo.
Nucleic acid modification enzymes, particularly DNA ligases and helicases, play a critical role in nucleic acid replication, excision repair, and genetic recombination. Ligases catalyze the ligation of nucleic acid strand breaks generated during various cellular metabolic events such as nucleic acid replication and recombination, and in response to strand breaks produced by various environmental factors. DNA helicases unwind DNA as part of the replication process, and to facilitate removal and substitution of single strand nucleotides. The reaction mechanism of nucleic acid ligases and helicases has mainly been studied using radioisotope-labeled substrates and gel electrophoresis. Such techniques are time consuming, require disposal of environmentally dangerous substances, and limit the number of samples that can be assayed. Therefore, there is a need for an assay that is quick and does not require use of radiolabels or gel electrophoresis.
Fluorescence polarization has also been used to analyze fluorescently labeled molecules and ligands. Representative samples of such methods are disclosed in U.S. Pat. No. 5,789,249, which discloses the detection of nucleic acid cleavage enzymes, such as restriction enzymes; U.S. Pat. No. 4,902,630, which discloses use of fluorescence polarization to measure the amount of tracer-antibody complex produced in a competitive binding immunoassay; U.S. Pat. No. 4,681,859, which describes an immunoassay for detecting the presence of a ligand in an aqueous sample based upon its competition with a fluorescently labeled ligand for the binding site on an anti-ligand partner; and U.S. Pat. No. 4,751,190, which discloses another fluorescence polarization method for use in immunoassays. However, the present art does not disclose detecting enzymes used in the processes of nucleic acid repair and replication using fluorescence polarization according to the method and kit of the present invention.
The method and kit of the present invention uses fluorescence polarization to identify, in a homogeneous format, inhibitors of nucleic acid modification enzyme activity, in particular, nucleic acid ligase and helicase activity. Nucleic acid ligase repairs single-strand breaks, whereas helicases are often referred to as relaxing enzymes, because they unwind double stranded nucleic acids prior to replication and nucleic acid repair. The method and kit of the present invention measure DNA ligase and helicase activity, as well as the effect of inhibitors on such activities, using fluorescence polarization.
In one embodiment of the present invention, nucleic acid ligases and their inhibitors are identified by providing a first oligo, a second oligo, and a third fluorescein-labeled oligo, wherein the second and third oligos are complementary to adjacent sections of the first oligo and are capable of annealing thereto. The oligos are annealed to form a double stranded enzyme substrate with a break or nick between the second and third oligos. The substrate of the present invention is then diluted to the appropriate concentration in a buffer that is compatible with nucleic acid ligase activity. A nucleic acid ligase, an inhibitor or test compound, and the substrate are added together to form a reaction mixture, which is incubated for a time sufficient for reactions to occur. A stop reagent, such as NaOH, is added to dissociate the oligos and fluorescence polarization detects whether the test compound repaired the break between the second and third oligos to form a longer fourth oligo. Increased fluorescence polarization relative to that of the fluorescein-labeled third oligo indicates a ligation product (fourth oligo) is formed and that the test compound did not inhibit the activity of the ligase. A fluorescence polarization corresponding to the fluorescence polarization signal of the third oligo indicates that the test compound inhibits enzyme activity.
In another embodiment of the present invention a substrate comprised of two oligos is used to identify inhibitors of nucleic acid ligases and helicases. One oligo includes a section that is complementary and anneals to the second oligo, which is fluorescently labeled. A break or nick is located between the adjacent ends of the two oligos. The entire structure or substrate forms a double stranded hairpin nucleic acid molecule, which is characterized by a particular fluorescence polarization signal. In the presence of a helicase, unwinding and separation of the small fluorescein-labeled second oligo from the larger unlabelled oligo occurs. In the presence of ligase, the break between the two oligo is repaired, thereby linking the two oligos together to form a longer fluorescent-labeled oligo. Relevant inhibitors, would of course, prevent or inhibit the formation of these enzymatic products. Due to the different degrees of polarization of emitted light from reaction mixtures, the invention detects the presence of the enzymes and relevant inhibitors.
The invention also includes a kit for analyzing the activity of nucleic acid ligases and helicases and inhibitors thereof. The kit contains substrates and enzymes, along with an assay support structure for carrying out the steps of the method.
The method of using fluorescence polarization to assay the activity of nucleic acid ligases and helicases is limited to neither the use of fluorescein as the fluorescent label, the use of the specific oligos or numbers thereof, the biotin/avidin used as ligands for affecting a change in mass of an oligonucleotide, or the set of annealed oligonucleotides used.
The methods and kits of the present invention can for example be used to screen for crop protection chemicals, such as herbicides, insecticides or fungicides, antibiotics, anti-viral agents, anti-tumor agents, or compounds, which modulate and/or regulate the function of clinically important proteins.
An advantage of the methods and kits of the present invention is that they are particularly well-suited for high-throughput assays.